The role of platelets in hemostasis is to arrest bleeding. An interaction between the integrin receptor GP JIb-lila (a11b133) and fibrinogen leads to platelet spreading, aggregation, and ultimately clot formation. These outcomes require a series of coordinated signaling events that involve protein tyrosine phosphorylation. A subset of proteins is not phosphorylated in platelets from Glanzmann's thrombosthenia patients that fail to aggregate or in normal platelets that do not spread. We hypothesize that this subset of proteins mediates GP Jib-lila dependent signals leading to the organization of the cytoskeleton and platelet aggregation and spreading. Experiments to test this hypothesis will focus on three proteins that we have found are phosphorylated in spread and/or aggregated platelets: a-actinin, vinculin, and a lO1-kD protein (pplOl) that is identical or related to VCP (Valosin-containing protein). Recently we reported that a-actinin is tyrosine phosphorylated in spread platelets. We obtained mass spectroscopy data suggesting that pplOl is identical or related to VCP, a 100-kD protein that is tyrosine phosphorylated in hematopoietic and non-hematopoietic cells. We also purified a protein of 1 30-kD that is tyrosine phosphorylated in spread and/or aggregated platelets. The protein was identified as vinculin by mass spectroscopy. We will address the following specific aims: Aim 1. What is the role of tyrosine phosphorylation of a-actinin? Aim 2: Which phosphatase(s) and kinase(s) regulate the state of phosphorylation of a-actinin? Aim 3: What is the role of tyrosine phosphorylation of pplOl and vinculin, and which phosphatase(s) and kinase(s) regulate these phosphorylation events?